This invention relates to a method for immunochemical assay of human chorionic gonadotropin (hereinafter briefly, hCG) and to production of an antibody usable for the assay.
hCG is a proteohormone produced from chorionic cells formed on conception, and stimulates secretion of progesterone. Detection of hCG is a technique commonly utilized as an early diagnostic procedure for pregnancy. Furthermore, in chorionic diseases such as hydatidiform mole, destructive mole, villous cancer, etc. determination of hCG in the urine, blood or other body fluid has proved to be very beneficial for an early detection of such disorders, evaluation of effects of treatments and prognostic management of the diseases. However, for diagnosis of these diseases, a detection sensitivity of less than about 100 IU/l of hCG is required and there is the problem of immunological cross-reactivity with other proteohormones structurally analogous to hCG, such as luteinizing hormone (hereinafter sometimes referred to as hLH), follicle stimulating hormone (hereinafter sometimes referred to as hFSH) and thyroid stimulating hormone (hereinafter sometimes referred to as hTSH). Among them, hLH, in particular, is very similar to hCG and the amount of hLH in physiological urine may at times be as high as 150 IU/l. Therefore, in order to measure hCG in the body fluid, it is necessary to distinguish hCG from hLH in an immunological sense.
On the other hand, chemical analyses of these proteohormones have shown that the above-mentioned cross-reactivity is due to their .alpha.-subunits which structurally have much in common. Therefore, the comparatively less analogous .beta.-subunit of hCG (hereinafter sometimes referred to as hCG-.beta.) was separated and purified and an anti-hCG-.beta. antibody was prepared and used for specific detection of hCG. However, the separation and purification of hCG-.beta. requires a complicated procedure and it is also very difficult to avoid contamination with hCG and the .alpha.-subunit of hCG (hereinafter sometimes referred to as hCG-.alpha.). The concomitant presence of these impurities and the still remaining common amino acid sequence of the .beta.-subunits of hCG and hLH do not allow the cross-reactivity with hLH to be eliminated completely with use of the anti-hCG-.beta. antibody. However, the peptide fragment consisting of about 30 amino acid residues at the C-terminal of hCG-.beta. has an amino acid sequence not found in hLH, and it was found that, in this particular moiety, hCG can be completely differentiated from hLH.
Based on these results of structural analysis, Matsuura et al. synthesized the C-terminal peptide of hCG-.beta., immunized rabbits with the peptide and carried out radioimmunoassays (hereafter briefly, RIA) with the resulting hCG-specific antiserum by the competitive method [Endocrinology, Vol. 104 (1979), P. 396]. Though they obtained satisfactory results as to specificity, the sensitivity obtained was not sufficient.
In European patent application No. 81,102,360.5, the anti-hCG antibody obtained by immunization of an animal with hCG is contacted with the solid phase obtained by immobilizing the synthetic C-terminal peptide of hCG-.beta., and the anti-hCG antibody specifically absorbed is used in competitive enzyme immunoassays (hereafter sometimes briefly referred to EIA) with satisfactory specificity and sensitivity. However, RIA and EIA by the competitive method are liable to be influenced by other components in test fluids and, as an additional disadvantage, require prolonged times for assays. Therefore, the present inventors sought a more expedient method of assay. The investigation led to the finding that hCG could be assayed with high specificity and sensitivity by the non-competitive method (hereafter referred to as the sandwich method). This invention has been accomplished on the basis of the above finding.